Characterization of Glycation Adducts On Human Serum Albumin by Matrix Assisted Laser Desorption/ Ionization Time-Of-Flight Mass Spectrometry

Background—Non-enzymatic glycation of human serum albumin (HSA) is associated with the long-term complications of diabetes. We examined the structure and location of modifications on minimally glycated HSA and considered their possible impact on the binding of drugs to this protein. Methods—Minimally glycated and normal HSA (used as a control) were digested with trypsin, Glu-C or Lys-C, followed by fractionation of the resulting peptides and their analysis by matrixassisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) to determine the structures and locations of glycation adducts. Results—Several specific lysine and arginine residues were identified as modification sites in minimally glycated HSA. Residues K12, K51, K199, K205, K439 and K538 were found to be modified through the formation of fructosyl-lysine, while the modification of K159 and K286 involved the formation of pyrraline, Nε-carboxymethyl-lysine respectively. Lysine K378 was found to give Nε-carboxyethyl-lysine in some forms of glycated HSA but fructosyl-lysine in other forms. Residues R160 and R472 produced a modification based on Nε-(5-hydro-4-imidazolon-2-yl) ornithine. Lysine R222 was modified to produce argpyrimidine, Nε-[5-(2,3,4-trihydroxybutyl)-5- hydro-4-imidazolon-2-yl]ornithine or tetrahydropyrimidine. Conclusions—With the exception of K12, K199, K378, K439 and K525, all of the observed sites of modification for minimally glycated HSA were new to this current study. The fact that many of these glycation-related modifications are located at or near known drug binding sites on HSA explains why some differences have been previously noted in the binding of certain drugs to normal vs glycated HSA

Characterization of affinity ligands by MALDI-TOF MS and the preparation of affinity restricted access media

The first topic considered in this dissertation was an investigation of heterogeneous protein ligands using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA) was used as a model in these studies. A means to obtain high sequence coverage in protein studies by MALDI-TOF MS was developed by optimizing matrix preparation and using multiple enzyme digests with peptide fractionation. The optimized method could be used to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein. This MALDI-TOF MS method was applied in quantitative studies of heterogeneous immobilization sites on HSA that had been coupled to silica through the Schiff base method. The immobilized HSA and soluble HSA were digested separately in normal water or 18O-enriched water and then mixed together for analysis by MALDI-TOF MS. Several peptides were found to have significantly higher 18O/16O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA. MALDI-TOF MS was also applied in studying the inherent heterogeneity in minimally glycated HSA. By comparing the peptide maps of normal HSA and glycated HSA, twelve lysines and three arginines were found to be likely modification sites in minimally glycated HSA. The modifications on these sites were also identified by employing their observed mass shifts. The second general topic of this dissertation focused on the development of affinity restricted access media using antibodies as immobilized ligands. Rabbit IgG and anti-fluorescein antibodies were used as model ligands in this work. These antibodies were first immobilized onto silica supports, with ligands on the outer surface being cleaved by an enzyme while antibodies in the support pores were left intact. Items evaluated in the development of such media included the immobilization method, pore size of support, the type of enzyme used support treatment, the levels of this enzyme level and the digestion time. The optimized conditions were used to prepare the anti-fluorescein antibodies restricted access media columns for ultrafast immunoextraction. Injections of fluorescein and fluorescein-labeled bovine serum albumin on this column indicated that the resulting stationary phase had high selectivity for small versus large molecules containing the same binding regions for antibodies

Identification and Quantitative Studies of Protein Immobilization Sites by Stable Isotope Labeling And Mass Spectrometry

A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in 18O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the 18O/16O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher 18O/16O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313 and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports

Development Of Sulfhydryl-Reactive Silica For Protein Immobilization In High-Performance Affinity Chromatography

Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in highperformance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77–81% of this protein to maleimide- or iodacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, non-specific binding, stability and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations

Restricted Access Media and Methods for Making Restricted Access Media

The present invention is directed to restricted access media (RAM), methods for preparing restricted access media, and kits for preparing restricted access media that contain protected ligand binding agents or protected enzymes. Certain RAM provided contain a plurality of protected regions of the Support that contain ligand binding agents that are protected by blocking agents. Certain RAM provided contain a plurality of protected regions of the support that contain unbound ligand binding agents or enzymes that are retained in the protected regions by a capping agent. Methods of making the RAM of the invention and associated kits are also provided

Biointeraction Analysis of Carbamazepine Binding To Alpha 1-Acid Glycoprotein by High-Performance Affinitychromatography

Interactions of the drug carbamazepine with the serum protein α1-acid glycoprotein (AGP) were examined by high-performance affinity chromatography (HPAC). Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low affinity interactions with the support and high affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (± 0.1) × 105 M−1, with values that ranged from 5.1 to 0.58 × 105 M−1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how HPAC can be used to examine biological interactions and drug-protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand

QUANTITATIVE ANALYSIS OF GLYCATION SITES ON HUMAN SERUM ALBUMIN USING 16O/18O-LABELING AND MATRIXASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY

Background—One of the long term complications of diabetes is the non-enzymatic addition of glucose to proteins in blood, such as human serum albumin (HSA), which leads to the formation of an Amadori product and advanced glycation end products (AGEs). This study uses 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to provide quantitative data on the extent of modification that occurs in the presence of glucose at various regions in the structure of minimally glycated HSA. Methods—Normal HSA, with no significant levels of glycation, was digested by various proteolytic enzymes in the presence of water, while a similar sample containing in vitro glycated HSA was digested in 18O-enriched water. These samples were then mixed and the 16O/18O ratios were measured for peptides in each digest. The values obtained for the 16O/18O ratios of the detected peptides for the mixed sample were used to determine the degree of modification that occurred in various regions of glycated HSA. Results—Peptides containing arginines 114, 81, or 218 and lysines 413, 432, 159, 212, or 323 were found to have 16O/18O ratios greater than a cut off value of 2.0 (i.e., a cut off value based on results noted when using only normal HSA as a reference). A qualitative comparison of the 16O- and 18Olabeled digests indicated that lysines 525 and 439 also had significant degrees of modification. The modifications that occurred at these sites were variations of fructosyl-lysine and AGEs which included 1-alkyl-2-formyl-3,4-glycoyl-pyrole, and pyrraline. Conclusions—Peptides containing arginine 218 and lysines 212, 413, 432, and 439 contained high levels of modification and are also present near the major drug binding sites on HSA. This result is clinically relevant because it suggests the glycation of HSA may alter its ability to bind various drugs and small solutes in blood

Review: Glycation of human serum albumin

Glycation involves the non-enzymatic addition of reducing sugars and/or their reactive degradation products to amine groups on proteins. This process is promoted by the presence of elevated blood glucose concentrations in diabetes and occurs with various proteins that include human serum albumin (HSA). This review examines work that has been conducted in the study and analysis of glycated HSA. The general structure and properties of HSA are discussed, along with the reactions that can lead to modification of this protein during glycation. The use of glycated HSA as a short-to-intermediate term marker for glycemic control in diabetes is examined, and approaches that have been utilized for measuring glycated HSA are summarized. Structural studies of glycated HSA are reviewed, as acquired for both in vivo and in vitro glycated HSA, along with data that have been obtained on the rate and thermodynamics of HSA glycation. In addition, this review considers various studies that have investigated the effects of glycation on the binding of HSA with drugs, fatty acids and other solutes and the potential clinical significance of these effects